Ribozymes are ideal model systems for the vast number of non-protein coding RNAs found in all domains of life, since they have an easily detectable biological function - catalysis. They also are of high biological and biotechnological relevance in their own right for their roles in the processing and regulation of genetic information. Yet, a quarter century after their discovery, our understanding of catalysis by ribozymes still pales compared to that of catalysis by protein enzymes. Over the last two funding cycles, the PI's group has made substantial contributions to our understanding of the folding and mechanism of the class of small ribozymes. All five members of this class were investigated to varying degrees, with particular focus on the hammerhead and hepatitis delta virus (HDV) ribozymes. Several important discoveries were also made on the hairpin ribozyme as a particularly intriguing model system, on which we will follow up during the current funding period, bringing to bear our signature integration of biophysical and biochemical tools. In Specific Aim 1, we will test the hypothesis that the persistent folding heterogeneity of the hairpin ribozyme, observed at the single molecule level, is caused by slow repuckering of specific nucleotide sugars. Similar folding heterogeneity of chemically identical isomers has been observed for a number of RNAs when (re)folded in vitro, but still lacks a molecular explanation. We have recently succeeded in avoiding this heterogeneity when natively purifying the RNA directly from an in vitro transcription reaction, paving the way for investigating the molecular basis of folding heterogeneity in the hairpin ribozyme by a combination of single molecule fluorescence resonance energy transfer (smFRET), footprinting, and molecular dynamics (MD) simulations. In Specific Aim 2, in collaboration with Jiri Sponer, a computational scientist and long-standing collaborator, and Joseph Wedekind, an X-ray crystallographer, we will test the hypothesis that a network of global molecular motions in the hairpin ribozyme has an impact on those local molecular motions that lead to catalysis. Such a linkage has been suggested for protein enzymes, but has not been rigorously tested for any ribozyme. To this end, we will introduce site-specific modifications into the hairpin ribozyme and probe, using a combination of enzymology, smFRET, X-ray crystallography, and MD simulation, the impact of each of these modifications on local and global structure, dynamics, and function. In Specific Aim 3, we will test a set of specific mechanistic proposals for the role of A38 and water in catalysis of the hairpin ribozyme. This aim follows up on our previous observation that a judiciously placed A38 residue is flanked in the solvent- protected catalytic core by several tightly bound water molecules. We will pursue a broadly sampled QM/MM treatment of the catalytic reaction in collaboration with Jiri Sponer and Joseph Wedekind, as well as quantum chemist Michal Otyepka. We anticipate that results from these three Specific Aims will significantly deepen our understanding of the biological function of non-coding RNAs in general.